Tetrairidium and undecagold clusters will be synthetically optimized and used for site-specific derivatization of membrane protein crystals within their hydrophobic domains, in order to assign phases for X-ray diffraction data during crystal structure determination. Tris (alkyl) or (aryl) phosphine ligands will be used to confer hydrophobicity and structural rigidity for compatibility with specialized membrane protein-solubilizing detergents. A small number of hydrophilic ligand substituents will be used to introduce asymmetric, partial affinities for the polar head-groups, specifically orienting the cluster within the detergent monolayer and limiting disorder in its atomic coordinates. Crystals of cytochrome bc1 complex will be derivatized using two methods: (i) reaction of maleimido- forms of the new clusters with cysteine residues; and (ii) conjugates of the clusters with 5- nitrosalicylic acid, an inhibitor, to target the cluster to the active site. Optimum wavelengths for multi-anomalous dispersion (MAD) phasing will be calculated from EXAFS measurements on the derivatized crystals. X-ray diffraction data will then be collected, refined, and combined with partial phases from other experiments to determine the phasing power and occupancy of the new clusters, and ultimately to solve the crystal structure of the cytochrome bc1 complex to atomic resolution. PROPOSED COMMERCIAL APPLICATION: Membrane proteins are fundamentally important in many cellular processes, and their structural characterization is the focus of intense interest. Although only a small number have been completely solved, the proposed unique class of derivatization reagents, synthetically designed to complement the novel detergents which have been developed specifically for membrane protein crystallization, will potentially enable the X-ray crystal structure determination of many more at up to atomic resolution.